Anja Wittmann, Benjamin Roenneke

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Scientific Background

Cyanobacteria are phototrophic prokaryotes, which inhabit environments of widely differering conditions in nearly all habitats on earth.
So they are able to survive in freshwater as well as in sea water by adjusting the water potential of the cytoplasm. While the components involved in this acclimatization are characterized (Fig. 1), the regulation of this process is poorly understood.
Sensing of salinity changes and fine tuning of de novo synthesis and uptake of compatible solutes in the moderate halotolerant model strain Synechocystis sp. PCC 6803 are topics we are interested in (see Fig. 2).
By the in vitro analysis of enzyme activities under defined conditions we can monitor the dependency on different parameters as well as the interaction of proteins by biochemical methods.
Additional regulatory factors can be identified by affinity chromatography with overexpressed proteins or by transposon mutagenesis and subsequent selection of positive clones.
The participation of proteases in osmoregulation is under investigation by the characterization of FtsH protease mutants.
Degradation of proteolytic substrates can be analysed in vivo by the 2D-Page technique and in vitro after heterologous production of putative substrates and incubation with inverted membrane vesicles containing different FtsH proteases or after reconstitution of the membrane bound proteases.
By the subsequent mutagenesis of proteases as well as target proteins the mechanisms of substrate recognition and degradation will be studied on the molecular level.

Fig. 2: Working model of the regulation of osmolyte synthesis in Synechocystis on the posttranslational level. After an initial salt stress dependent activation the GgpS protein forms a complex with the second enzyme of the pathway GgpP, which results in GG-synthesis. By a so far unknown mechanism the GgpS protein becomes inactivated and the membrane bound FtH2 protease is responsible for the removal and subsequent degradation of the inactive GgpS protein. After liberation of the GgpP enzyme it can form a new complex with active GgpS protein.

Aims

Identification of mechanisms involved in stress dependent activation and inactivation of enzyme activities during the osmotic acclimatization process in Synechocystis.
Understanding of interaction of proteolysis and osmoregulation on the molecular level.
Identification and characterization of new components involved in regulatory principles and pathways and verification of occurence in other cyanobacteria or eubacteria.

Projects

Steady state activity regulation of the key enzyme of osmolyte synthesis in Synechocystis

In all living cells the water content of the cytoplasm has to be adjusted in relation to the external medium osmolality.
In Synechocystis the synthesis and accumulation of the osmoprotective compound glucosylglycerol (GG) is an essential process during osmotic acclimatization.
GG synthesis is performed by the enzymes Glucosylglycerol-phosphate-synthase (GGPS), which is the key enzyme and Glucosylglycerol-phosphate-phosphatase (GGPP).
These enzymes might form a complex after the release of the GgpS enzyme from a postulated inhibitor (A).
During steady state regulation most of the enzyme is inactivated by a so far unknown mechanism (A), but can be reactivated rapidly by an increase of the external salt concentration.
We aim to understand the mechanism of GG synthesis regulation by searching for involved proteins by transposon mutagenesis and screening for clones with deregulated GG synthesis.
By heterologous production of the involved enzymes (B) we analyze activation and inactivation by in vitro enzyme assays and focus also on enzyme kinetics and structure.

The proposed model for GG synthesis regulation (A) includes the release of the GgpS enzyme from an inhibitor and the interaction with the GgpP enzyme during GG synthesis.
By a so far unknown mechanism the complex is inactivated but can be reactivated after an increase of the external salt concentration.
For in vitro investigation involved components are heterologously produced (B) and analyzed by enzyme assays under different conditions (B).
The structure function relation of the GgpS enzyme can be visualized by the proposed structure model.

Interplay between protein quality control and osmoregulation in Synechocystis

Protein quality control and degradation of misfolded and irreversibly inactivated enzymes is an essential process in all living cells.
A ubiquitous proteolytic machinery is the membrane bound ATP and Zn dependent FtsH protease complex, constituted of six FtsH proteins.
While in heterotrophic bacteria only one gene encoding an FtsH protease is present cyanobacteria possesses four different FtsH proteins.
We have identified the FtsH2 protease of Synechocystis as a player in osmoregulation responsible for the degradation of irreversibly inactivated GgpS enzyme.
This is an essential procedure because the ftsH2 mutant is impaired in GG synthesis and therefore salt sensitive (A).
The proteolytic degradation of GgpS by FtsH2 was proven in an in vitro assay using inverted membrane vesicles and heterologously produced GgpS protein (C).
Besides GgpS further putative soluble substrates were identified by 2D-PAGE analysis (B).
We aim to unravel the mechanism of substrate recognition on the molecular level and thereby to address the questions

how the FtsH2 protease can discriminate between the active and inactive GgpS protein,

what makes the inactive GgpS protein a substrate of the protease and

why only FtsH2 is involved in GgpS degradation while the other three FtsH proteases are not.

In Synechocystis four FtsH proteases are present and FtsH2 was found to be involved in degradation of the GgpS protein.
Besides the known FtsH2 targets the D1 protein and GgpS other soluble proteins were identified by 2D-PAGE (B) as putative substrates of the proteolytic machinery because of their accumulation in the ftsH2- mutant.
After heterologous production and purification of substrates the degradation can be analyzed in vitro (C).

Methods

By a combination of physiological and biochemical approaches we want to understand regulatory mechanisms on the molecular level.

Physiological methods

Besides the cultivation of Synechocystis on agar plates or in Erlenmeyer flasks (A) growth with elevated CO2 concentrations can be performed (B).
Cell size and shape under osmotic stress conditions can be analyzed by light (C) or fluorescence microscopy.

Molecular methods

For generation of mutants we use insertions of antibiotic resistance markers (A) or deletions of genes (B). For complementation of mutants plasmid encoded genes can be used.
For screening procedures random mutant collections are generated by transposon mutagenesis (D).

All routine molecular biology techniques for cloning and modification of genes (A) are established.
Several heterologous expression systems are in use (B).
Gene expression can be monitored by Northern-Blot as well as Western-Blot techniques (C) and in collaboration with other groups by DNA microarray (D) or 2D-PAGE analysis (E).

Biochemical methods

For identification of regulatory compounds and quantification of external and internal substrate concentration we use HPLC, GC as well as GC-MS.
By the time dependent substrate concentration analysis the kinetic parameters of the transport process can be determined.

Spectroscopic methods are in use for the analysis of pigmentation (A) the efficiency of photosystems as well as protein conformations (C) and protein protein interactions.
Data can be visualized by computer based structure predictions (C) in order to design new modifications, e.g. by mutagenesis.